Evolutionary conservation of the structure and function of meiotic Rec114-Mei4 and Mer2 complexes.

Meiosis-specific Rec114-Mei4 and Mer2 complexes are thought to enable Spo11-mediated DNA double-strand break (DSB) formation through a mechanism that involves DNA-dependent condensation. However, the structure, molecular properties, and evolutionary conservation of Rec114-Mei4 and Mer2 are unclear. Here, we present AlphaFold models of Rec114-Mei4 and Mer2 complexes supported by nuclear magnetic resonance (NMR) spectroscopy, small-angle X-ray scattering (SAXS), and mutagenesis. We show that dimers composed of the Rec114 C terminus form α-helical chains that cup an N-terminal Mei4 α helix, and that Mer2 forms a parallel homotetrameric coiled coil. Both Rec114-Mei4 and Mer2 bind preferentially to branched DNA substrates, indicative of multivalent protein-DNA interactions. Indeed, the Rec114-Mei4 interaction domain contains two DNA-binding sites that point in opposite directions and drive condensation. The Mer2 coiled-coil domain bridges coaligned DNA duplexes, likely through extensive electrostatic interactions along the length of the coiled coil. Finally, we show that the structures of Rec114-Mei4 and Mer2 are conserved across eukaryotes, while DNA-binding properties vary significantly. This work provides insights into the mechanism whereby Rec114-Mei4 and Mer2 complexes promote the assembly of the meiotic DSB machinery and suggests a model in which Mer2 condensation is the essential driver of assembly, with the DNA-binding activity of Rec114-Mei4 playing a supportive role.

Cosolvent Molecular Dynamics Applied to DPP4, DPP8 and DPP9: Reproduction of Important Binding Features and Use in Inhibitor Design.

We present our efforts in computational drug design against dipeptidyl peptidase 4 (DPP4), DPP8 and DPP9. We applied cosolvent molecular dynamics (MD) simulations to these three protein targets of interest. Our primary motivation is the growing interest in DPP8 and DPP9 as emerging drug targets. Due to the high similarity between DPP4, DPP8 and DPP9, DPP4 was also included in these analyses. The cosolvent molecular dynamics simulations reproduce key ligand binding features and known binding pockets, while also highlighting interesting fragment positions for future ligand optimization. The resulting fragment maps from the cosolvent molecular dynamics are freely available for use in future research (https://github.com/UAMC-Olivier/DPP489_cosolvent_MD/). Detailed instructions for easy visualization of the fragment maps are provided, ensuring that the results are usable by both computational and medicinal chemists. Additionally, we used the fragment maps to search for the binding pockets with significant potential using an algorithmic approach combining top fragment locations. To discover novel binding scaffolds, a limited pharmacophore screening was performed, where the pharmacophores were based on the analyses of the cosolvent simulations. Unfortunately, inhibitory potencies were in the higher micromolar range, but we optimized the resulting scaffolds in silico using relative binding free energy calculations for future inhibitor design and synthesis.

Active site-directed probes targeting dipeptidyl peptidases 8 and 9.

Dipeptidyl peptidases (DPP) 8 and 9 are intracellular serine proteases that play key roles in various biological processes and recent findings highlight DPP8 and DPP9 as potential therapeutic targets for hematological and inflammasome-related diseases. Despite the substantial progress, the precise biological functions of these proteases remain elusive, and the lack of selective chemical tools hampers ongoing research. In this paper, we describe the synthesis and biochemical evaluation of the first active site-directed DPP8/9 probes which are derived from DPP8/9 inhibitors developed in-house. Specifically, we synthesized fluorescent inhibitors containing nitrobenzoxadiazole (NBD), dansyl (DNS) and cyanine-3 (Cy3) reporters to visualize intracellular DPP8/9. We demonstrate that the fluorescent inhibitors have high affinity and selectivity towards DPP8/9 over related S9 family members. The NBD-labeled DPP8/9 inhibitors were nominated as the best in class compounds to visualize DPP8/9 in human cells. Furthermore, a method has been developed for selective labeling and visualization of active DPP8/9 in vitro by fluorescence microscopy. A collection of potent and selective biotinylated DPP8/9-targeting probes was also prepared by replacing the fluorescent reporter with a biotin group. The present work provides the first DPP8/9-targeting fluorescent compounds as useful chemical tools for the study of DPP8 and DPP9's biological functions.

Biophysical analysis of the membrane-proximal Venus Flytrap domain of ESAG4 receptor-like adenylate cyclase from Trypanosoma brucei.

The protozoan parasite Trypanosoma brucei possesses a large family of transmembrane receptor-like adenylate cyclases (RACs), primarily located to the flagellar surface and involved in sensing of the extracellular environment. RACs exhibit a conserved topology characterized by a large N-terminal extracellular moiety harbouring two Venus Flytrap (VFT) bilobate structures separated from an intracellular catalytic domain by a single transmembrane helix. RAC activation, which typically occurs under mild acid stress, requires the dimerization of the intracellular catalytic domain. The occurrence of VFT domains in the RAC's extracellular moiety suggests their potential responsiveness to extracellular ligands in the absence of stress, although no such ligands have been identified so far. Herein we report the biophysical characterization of the membrane-proximal VFT2 domain of a bloodstream form-specific RAC called ESAG4, whose ectodomain 3D structure is completely unknown. The paper describes an AlphaFold2-based optimisation of the expression construct, enabling facile and high-yield recombinant production and purification of the target protein. Through an interdisciplinary approach combining various biophysical methods, we demonstrate that the optimised VFT2 domain obtained by recombination is properly folded and behaves as a monomer in solution. The latter suggests a ligand-binding capacity independent of dimerization, unlike typical mammalian VFT receptors, as guanylate cyclase. In silico VFT2 genomic analyses shows divergence among cyclase isoforms, hinting at ligand specificity. Taken together this improved procedure enabling facile and high-yield recombinant production and purification of the target protein could benefit researchers studying trypanosomal RAC VFT domains but also any trypanosome domain with poorly defined boundaries. Additionally, our findings support the stable monomeric VFT2 domain as a useful tool for future structural investigations and ligand screening.